This invention concerns biomedical assays in which light is emitted by a light emitting component (if present), usually triggered by excitation.
In such assays, the existence of the light emitting component is determined by detecting the emitted light using a sensitive light detecting device such as a cooled CCD detector or the like.
The wavelength of the emitted light is unique to the component and by using filters etc. the presence of other wavelengths can theoretically be masked from the detector. However, for various reasons, significant quantities of light at other wavelengths can reach the detector, during the light detection mode.
Thus, in assay systems in which a sample or samples are emitting light, the minimal detectable light signal is governed by the light arising from various sources of background in the assay matrix and detection system. Accurate and precise measurement of background as well as of signal is a crucial step in determining the sensitivity of the assay. Such assay detection methods include fluorescence by epi- and trans-illumination; luminescence, including chemi- and bio-luminescence; and radioisotopic methods.
Variations in the efficiency of detection of light from such assays, particularly resulting from uncertainty in the position of the sample or samples with respect to the detector input, from lateral or vertical displacements and from angular displacement or orientation, also contribute to errors in measurement.
Again, scatter and reflections of excitation light from the sample matrix make a significant contribution to background in the detection system, and hence to the uncertainty of error in the measurement of background.
Typical matrices for sample presentation include the following: a multi-well plate (e.g. a microplate); a dish or tray; a membrane; a gel; a glass or silicon wafer type presentation substrate; a cuvette or a capillary or an array of such. Variations in the sample presentation matrix itself which can give rise to detection errors include non-flatness, curvature, ripples or dimples, undulations, variations in the thickness, transparency or reflectivity of the material of the matrix, any or all of which can modify the amount of light reaching the detector. Examples of this include the transparent base of a multi-well plate or dish, both across the plate as a whole and within the area of the well itself; membranes which can have undulations and thickness variations; and sample presentation substrates which can have variations in reflectivity.
The present invention seeks to allow the contribution of the light received by the detector from these variable sources of background to be quantified on an assay by assay basis, thereby allowing greater statistical certainty to be placed on the output signals from the detector when the assay is illuminated with excitation wavelengths, in the detection mode.
According to one aspect of the present invention there is provided a method of performing a biomedical assay, comprising the steps of:
exciting the sample or samples with incident radiation of a given wavelength, thereby causing the sample to emit radiation of a different wavelength,
measuring the quantity of light falling on a detector receiving the emitted radiation from the sample, thereby to produce a first measurement,
illuminating the sample or samples with incident radiation in a manner which does not cause the sample or samples to emit any significant radiation,
again detecting the quantity of light falling on the detector, thereby to produce a second measurement, and
correcting the first measurement with respect to the second measurement.
The first and second measurements may be made in either order.
In a less preferred method, in order to produce the second measurement, a light emission inhibitor is added to the sample or samples, and the sample or samples are illuminated with the same wavelength of radiation used to excite the sample or samples in order to obtain the first measurement.
In a preferred method, in order to produce the second measurement, the sample or samples are illuminated with a wavelength of light selected not to give rise to any significant emission of light from the sample or samples. The light of selected wavelength may lie in the infra-red or the ultra-violet region.
According to another aspect of the invention, there is provided apparatus for performing a biomedical assay in which light is emitted from a sample or samples when excited by incident radiation, comprising:
means for illuminating the sample or samples with incident radiation of two differing wavelengths,
detection means for detecting the radiation emitted by the sample when illuminated at the two wavelengths, thereby to produce a first and second measurement, and
means for correcting one measurement with respect to the other measurement.